In the case where a colorless transparent phase sample such as a biological cell is desired to be observed using an optical microscope, the structure cannot be seen by using a bright-field view imaging technique. However, various imaging techniques are known that make such a phase sample visible. Examples of these are the phase contrast imaging technique, the modulation contrast imaging technique, the differential interference contrast (DIC) imaging technique, and so on.
The phase contrast imaging technique positions a ring ("ring" herein means--annular shape--) slit at a pupil plane of an illuminating optical system of a microscope, and arranges a phase-shifter (i.e., a layer which provides phase contrast) at a conjugate pupil plane of an image-forming optical system of the microscope. The relative phase delay of the light diffracted by the phase structure of the sample is only .pi./2 radians as compared to the zero-order light. The phase-shifter is positioned at the region where the zero-order light passes in order to negate the phase difference between the zero-order light and the diffracting light. Thus, an image of the phase structure becomes visible.
In general, light attenuation of an appropriate amount is provided on the phase-shifter, and the contrast in the image is thus increased by having the zero-order light intensity and the light intensity of the diffracted light carefully controlled. This has the advantage that an image with distinctive contrast can be observed with high detection sensitivity even for minute structures such as the granular shapes inside of cells. On the other hand, a disadvantage is that the ends of the structures are seen as shining white, due to a phenomenon called halo, and thus the detection of the outline of the structures becomes difficult.
On the one hand, the modulation contrast imaging technique arranges an aperture slit at a pupil plane of an illuminating optical system of the microscope, as indicated in Japanese Patent Publication 51-128548, and arranges multiple regions with differing transmittances on an optical modulation element positioned at a conjugate pupil plane of an image-forming optical system of the microscope. Usually, a light absorbing layer is provided so that it has an appropriate transmittance at the conjugate region to the aperture slit. A region adjacent one side of this conjugate region is made to be a light transmitting region and the region on the other side is made to be a shaded (i.e., light blocking) region. In the pupil plane, the position where light from the slit passes varies depending on the structure in the sample so that the phase structure can be observed with shading. Advantages of the modulation contrast imaging technique are that an image having a three-dimensional sense is obtained and little expense is incurred in practicing this imaging technique. Also, this imaging technique is well-suited for manipulating cells and the like in that one can easily view the outline of the structures with no halo. On the other hand, a disadvantage of the modulation contrast imaging technique is that the discerning of minute structures is difficult, and that the detection sensitivity is inferior in contrast to the phase contrast imaging technique. In addition, whenever the objective lens is exchanged, a complicated operation must be done in order to align the aperture slit and the conjugate region thereto of the optical modulation element (i.e., the absorbing layer).
In the DIC imaging technique, a sample is illuminated by two polarized light beams having respective polarizations which are orthogonal to each other by using a birefringent crystal. This technique allows minute structures of the samples to be viewed as a result of the minute structures affecting the polarization of the light. The polarized light from the samples is made to interfere. The advantage of the DIC imaging technique is that a three-dimensional sense is rendered to the image having extremely high contrast. On the other hand, a disadvantage is that the equipment becomes expensive as a result of using the birefringent crystals. And, in the case where the sample itself or a substrate supporting the sample affects the polarization of light, an accurate image can not be obtained. For example, the DIC imaging technique is not generally suitable for observing samples through plastic surfaces because plastic can itself change the polarization of the light.
As explained above, in each prior art technique for imaging phase samples there are disadvantages that arise in addition to the advantages. Thus, an imaging technique is desired to avoid the disadvantages while attaining the advantages.
Especially in the case where examining and manipulating biological samples and the like are performed under a microscope, it is necessary to detect even transparent, minute structures with a high sensitivity. Moreover, in order to perform accurate manipulation of minute objects and the like, the outlines of the structures must be clearly visible. Also, it is important to be able to perform observations without the influence of the characteristic on polarization of samples or substrates. Further, there is a strong need to eliminate adjusting operations on complex optical systems arising from exchanging objectives of a microscope, as is frequently the case while observing microscopic structures. Thus, it is desirable to be able to detect and manipulate samples easily and efficiently.
There have been attempts in the prior art to achieve the above goals, in an inexpensive manner, by combining aspects of the prior art modulation contrast technique and the phase contrast technique. An example of such an attempt is given below.
In an embodiment of Japanese Patent Publication 57-178212, a slit aperture is arranged at a pupil plane in a position displaced outward from the optical axis and oriented normal to the direction of displacement from the optical axis. A light absorbing, phase-shifter having an appropriate transmittance is provided on an optical modulation element that is located at a pupil plane which is conjugate to this aperture. Also, as an another embodiment, among the regions adjoining the light absorbing, phase-shifter, the transmittance drops in at least one of the adjoining regions in the direction outward from the optical axis. In both of the embodiments, one of the positive/negative first-order diffracted light components is blocked. Thus, diffracted light that contributes to the image is allowed to pass on only one side of the light absorbing, phase-shifter which passes the zero-order light. In general, the contrast of the image using this technique drops as a result of the diffracted light on one side region in relation to the zero-order light being entirely blocked. For example, there is consideration of a thin flat phase object where the phase distribution is a sine wave with the period 1/p. The equiphase wave surface .PHI.(x) of the light that passed through this object can be represented as follows EQU .PHI.(x)=A cos (2 .pi. p x)
wherein,
x is the axis direction parallel to the object, and A is the amplitude of the phase distribution. Then, the complex amplitude distribution E(x) on the phase object can be considered: EQU E(x)=exp {i .PHI.(x)}
For a small phase change, i.e., when A&lt;&lt;1, ##EQU1##
The above first term is the zero-order light, the second term is the positive first-order light, and the third term is the negative first-order light. When the phase difference of .pi./2 radians between the zero-order versus the positive/negative first-order light is negated using a phase-shifter, the above equation becomes the below equivalent value E'(x). EQU E'(x)=1+(A/2) exp (2 .pi. i p x)+(A/2) exp (-2 .pi. i p x) Equation (1)
In equation (1), when the negative first order light is blocked: EQU E"(x)=1+(A/2) exp (2 .pi. i p x) Equation (2)
The respective intensities of equations (1) and (2) are given as I' (x) and II" (x), as indicated below. EQU I'(x)=.vertline.E'(x).vertline..sup.2 .congruent.1+2 A cos (2 .pi. p x) Equation (3) EQU I"(x)=.vertline.E"(x).vertline..sup.2 .congruent.1+A cos (2 .pi. p x) Equation (4)
Therefore, in comparing the blocking of one of the positive/negative first order light components from contributing to the image (as in equation (4)), to the case where there is no blocking of these light components (as in equation (3)), the contrast drops to approximately one-half. Thus, based on the structure indicated in Japanese Patent Publication 57-178212, even though modulation contrast imaging occurs, the advantage of a high detecting sensitivity as obtained in the phase contrast imaging technique is not obtained.
In general, in the modulation contrast imaging technique, relative to the direct light (i.e., the non-refracted light by a sample), the transmittance drops in all the light that is passed to one side, since a light absorbing layer is arranged to attenuate this light. This provides contrast so as to allow a phase object to be viewed, with the contrast having the difference of brightness in the direction of refraction by the sample. In Japanese Patent Publication 51-128548, an example of a transmittance distribution of the light absorbing region on an optical modulation element which is used by the modulation contrast imaging technique is shown. The region that the direct (i.e., zero order) light passes through on the optical modulation element is at the center. A region on one side adjoining this region has low transmittance. In such cases, even though there is contrast in an image of the phase object, the resolving power drops as a result of about half of the light flux that passes through the pupil plane being obstructed and thus not contributing to the image. In order to avoid decreasing the resolving power, in general, apertures are arranged displaced outward from the optical axis and the regions having low transmittance are narrowed. By said patent, each region on the optical modulation elements can have a phase shift effect. However, also in this case, as a result of the diffracted light being obstructed as compared to the zero-order light, the efficacy of the phase contrast technique is low, just like that of previously mentioned Japanese Patent Publication 57-178212.
Furthermore, in the above cases, because of having an asymmetric region on an optical modulation element in relation to the optical axis, every time the objective lens is exchanged, an adjusting operation must be done in order to match the alignment of the aperture and the optical modulation element. Specifically, in the case where the optical modulation element is arranged at a pupil plane interior of the objective lens, because the alignment of the optical modulation element is not necessarily uniform in a fixed direction with respect to each objective lens when the objective lens is attached on the revolver, the aperture must be realigned to each objective lens when the objective lens is exchanged. Also, along with changing the objective lens, the aperture must also be changed since the required aperture differs based on the magnification of the lens. In using a microscope the magnification is frequently switched, and every time this occurs an exchange operation of the aperture is required.
In U.S. Pat. No. 4,407,569, a phase shift region and a light absorbing region are independently prepared for selective insertion at a pupil plane of the image-forming optical system and, based on a suitable exchanging of the apertures that respectively correspond, one can selectively switch between the phase contrast image and the modulation contrast image. Therefore, both the phase contrast effect and the modulation contrast effect cannot be realized at the same time. In one embodiment of this patent, it is also disclosed that there are two respective apertures, one for phase contrast imaging and one for modulation contrast imaging, on the same element. However, based on this construction, there is a difficulty in simultaneously imaging using both techniques. The reason is that the zero-order light from the aperture for phase contrast imaging receives a phase shift, although the zero-order light from the aperture for modulation contrast imaging does not have such a phase shift. Therefore, the interference effect of the zero-order light and the diffracted light is diminished and the contrast in the image is reduced. Even if the light absorbing regions for modulation contrast imaging were given a phase shift as well, the interference effect between the zero-order light and the diffracted light would be less than desirable as a result of expanding the region in which the diffracted light is given a phase shift.
On the one hand, in order to control the characteristic halo in the phase contrast imaging technique, a phase contrast microscope that employs a light absorbing region apart from the phase shifter at a pupil plane is described in Japanese Patent Publication 8-94936. However, the obtaining of the effect of the modulation contrast by said phase contrast microscope is not shown. In fact, because the shape of the aperture is also restricted to a ring, a modulation contrast image is not obtained. Also, even where nothing is clearly shown specifically concerning the position and width, etc., of the light absorbing regions which are arranged on optical modulation elements, the effects of both the phase contrast image and the modulation contrast image cannot be simultaneously obtained based on Japanese Patent Publication No. 8-94936. In summary, both the three-dimensional sense of the modulation contrast imaging technique and the high detection sensitivity of the phase contrast imaging technique were not able to be simultaneously attained by techniques that were suggested in the prior art.
As an example that reduces the exchanging operation of the aperture accompanying the changing of the objective lens, a ring slit of Leica is given. As for the ring slit, the exchanging of the ring slit is not necessary when there are changes of the respective magnifications because the ring slit corresponds to magnifications of 10.times. to 40.times.. However, because the aperture shape is a ring, when there is use of a high magnification objective lens, the effective numerical aperture on the illuminating side of the microscope is small, and the resolving power drops. Also, the halo becomes stronger and it becomes difficult to detect the outline of the structure of the samples.
On the one hand, in order to increase the resolution upon enlarging the illuminating ring slit diameter when a low magnification objective lens is used, the region where the zero-order light passes through the pupil plane of the image-forming optical system becomes larger. Because of this, there is a problem of diminished contrast.